WO2007045247A2 - Peptides derives d'un recepteur du facteur de croissance des fibroblastes - Google Patents

Peptides derives d'un recepteur du facteur de croissance des fibroblastes Download PDF

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WO2007045247A2
WO2007045247A2 PCT/DK2006/000585 DK2006000585W WO2007045247A2 WO 2007045247 A2 WO2007045247 A2 WO 2007045247A2 DK 2006000585 W DK2006000585 W DK 2006000585W WO 2007045247 A2 WO2007045247 A2 WO 2007045247A2
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amino acid
seq
fgfr
peptide
peptide according
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PCT/DK2006/000585
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WO2007045247A3 (fr
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Vladimir Berezin
Vladislav V. Kiselyov
Elisabeth Bock
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Enkam Pharmaceuticals A/S
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Priority to EP06791473A priority Critical patent/EP1940875A2/fr
Priority to JP2008535889A priority patent/JP2009511614A/ja
Publication of WO2007045247A2 publication Critical patent/WO2007045247A2/fr
Publication of WO2007045247A3 publication Critical patent/WO2007045247A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/20—Hypnotics; Sedatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel peptides that are capable of binding to a receptor of the fibroblast growth factor receptor family (FGFRs) and modulating activity of said receptor.
  • the peptides of the invention are peptide fragments of FGFRs and derived from a binding site for the reciprocal binding of the immunoglobulin-like modules 1 and 2 of FGFR.
  • the invention discloses amino acid sequences derived from the reciprocal Ig1-to-lg2 binding site of FGFR and relates to use of peptides comprising said amino acid sequences and pharmaceutical compositions comprising thereof for the treatment of different pathological conditions, wherein FGFRs play a prominent role.
  • Fibroblast growth factor receptors are a family of closely related transmembrane tyrosine kinases (FGFR1 - FGFR4). FGFR activation and signalling are dependent on dimerization of the receptor which is induced by binding of FGFR natural ligand of the fibroblast growth factor family (FGFs), and it also requires participation of cell surface heparin or heparan sulphate proteoglycans (McKeehan et al., 1998; ltoh and Ornitz, 2004).
  • FGFs fibroblast growth factor family
  • FGFs FGF1-FGF23
  • FGFRs constitute an elaborate signaling system that participates in many developmental and repair processes of virtually all mammalian tissues (Bottcher and Niehrs, 2005), in particular, they play a prominent role in functioning of the peripheral and central neural system in the adult (of 23 members of the FGF family, ten have been identified in the brain (Jungnickel et al, (2004) MoI Cell Neurosci. 25:21-9; Reuss and von Bohlen und Halbach (2003) Cell Tissue Res. 313:139-57)).
  • the prototypical FGFR consists of three immunoglobulin-like modules (Ig1 - Ig3), a trans-membrane domain and a cytoplasmic tyrosine kinase domain.
  • the linker region between the Ig1 and Ig2 modules is very long, consisting of 20-30 amino acid residues, including a stretch of acidic amino acids termed the acid box.
  • FGFRs also bind heparin/heparan sulphate, which is required for the high-affinity FGF-FGFR interaction (Yayon et al., 1991 ; Ornitz et al., 1992).
  • the structure of the Ig 1 module is unknown and the physiological significance of the module is not well elucidated.
  • the Ig1 module is dispensable for FGF- FGFR binding
  • the triple Ig-module form of FGFR1 (FGFRI ⁇ ) has an 8- and 3-fold lower affinity for FGF1 and heparin, respectively, compared to the double Ig-module form (FGFRI ⁇ ) (Wang et al., 1995b).
  • NCAM has recently been regarded as a member of a new class of putative alternative ligands of FGFR, low affinity ligands (Kiselyov et al., 2003; Kiselyov et al. (2005) J Neurochem 94:1169-1179). There has been obtained evidence for a direct interac- tion between NCAM and the receptor and stimulation of FGFR by NCAM (Kiselyov et al. (2003) Structure (Camb) 11 :691-701 ).
  • NCAM fragment having the sequence EVYWAENQQGKSKA (FGL peptide) involved in the interaction between NCAM and FGFR has been suggested as a new candidate drug for the treatment of a variety of pathologic conditions where the activity of FGFR may play a role (WO 03/016351).
  • WO 03/016351 describes some biological effects of the FGL peptide due to binding and activating FGFR.
  • the present invention provides new compounds which may be advantageously used for modulating FGFR activity and FGFR-dependent biological processes, and thus the invention provides new candidate drugs for the treatment of FGFR-related diseases.
  • the present invention describes a novel FGFR binding site for the reciprocal binding of FGFR Ig1 and Ig2 modules of same FGFR molecule and discloses peptide sequences that are capable of binding to said binding site and thereby modulate the receptor activation.
  • the invention relates to novel peptide sequences capable of binding to FGFR and modulating FGFR activity.
  • a peptide sequence which is capable of binding to the reciprocal Ig1-to-lg2 binding site comprises the amino acid motif Q/E-(x) 3 -x p , wherein x p is a hydrophobic amino acid residue and
  • (x) c is an amino acid sequence of three amino acid resides, wherein x is any amino acid residue, or amino acid motif K/R-x p -(x) o- i-K/R, wherein x p is a hydrophobic amino acid residue, and (x) is a charged amino acid residue.
  • a peptide of the invention comprises of about 25 amino acid residues and comprises a fragment of FGFR derived from the reciprocal Ig1-to-lg2 binding site.
  • the invention discloses amino acid sequences derived from the reciprocal binding site of the invention and relates to pharmaceutical compositions comprising thereof.
  • Invention also relates to uses of the peptides and pharmaceutical compositions comprising thereof for the treatment of different pathological conditions, wherein FGFRs play a role in pathology and/or recovery from disease, for example for a) treatment of conditions of the central and peripheral nervous system associated with postoperative nerve damage, traumatic nerve damage, impaired myelina- tion of nerve fibers, postischaemic damage, e.g.
  • Parkinson's disease Alzheimer's disease, Huntington's disease, dementias such as multiinfarct dementia, sclerosis, nerve degeneration associated with diabetes mellitus, disorders affecting the circadian clock or neuro-muscular transmission, and schizophrenia, mood disorders, such as manic depression
  • d) prevention of death of heart muscle cells such as after acute myocardial infarction
  • FIG. 1 Structure of the FGFR1 Ig1 module.
  • FIG. 1 Binding of the FGFR1 Ig1 module to the combined FGFR Ig2-lg3 modules.
  • A) A plot of the equilibrium binding level of the Ig1 module to the lg2-3 modules versus the concentration of the Ig 1 module is shown. The binding was studied by means of SPR analysis. The Ig1 module was injected into the sensor chip at the specified concentrations. The binding is given as a response difference between the binding to the sensor chip with the immobilized lg2-3 modules and a blank sensor chip (unspecific binding). The binding is given as an average of six replicates, with the error bar showing standard deviations. The data were fitted with the theoretical curve in order to calculate the dissociation constant (Kd).
  • Figure 3 Mapping of the various FGFRI-ligand binding sites onto the structures of the FGFR1 Ig1 and Ig2 modules.
  • FIG. 4 Quantitative analysis of the auto-inhibitory effect of the FGFR1 Ig1 module.
  • A) The structure of FGFRI ⁇ (triple Ig form) is depicted schematically with various random conformations of the Ig1-lg2 linker (assuming that there is no interaction between the Ig 1 and Ig2 modules). The circle corresponds to the average distance between the N-termini of the FGFR1 Ig1 and Ig2 modules.
  • FIG. 5 Effect of the FGFR1 Ig1 and Ig2 modules and the derived peptides on phosphorylation of FGFR1.
  • TREX-293 cells stably transfected with FGFR1 containing a C-terminal Strepll-tag, were stimulated for 20 min with either 100 ng/ml FGF1, or other compounds at the specified concentrations.
  • FRD1 stands for the Ig1 module, FRD2 - the Ig2 module, FRDIa - peptide corresponding to the Ig1-lg2 binding site in the Ig 1 module, FRD2a and FRD2b - peptides corresponding to the Ig1-lg2 binding site in the Ig2 module.
  • FGFR was immunopurified using anti-phosphotyrosine antibodies and then analyzed by immunoblotting using antibodies against the Strepll-tag (Panel A). Quantification of FGFR1 phosphorylation (Panel B) was performed by densitometric analysis of the band intensity. Phosphorylation was estimated relative to the control (untreated cells), which was normalized to 100%. Error bar represents one standard error of the mean. P ⁇ 0.05 (marked by * ) and P ⁇ 0.01 (marked by ** ) by paired t-test when comparing treated cells with controls (the t-test was performed on six independent sets of non-normalized data).
  • Ig2 modules / is the linker length
  • p is the length of the Ig 1 module
  • ⁇ is the angle between a long axis of the Ig1 module and the straight line connecting the N terminal of the Ig2 module and the C terminal of the Ig1 module.
  • FIG. 7 Neurite outgrowth of cerebellar granular neurons in response to treatment with the peptides FRD2a (SEQ ID NO:8 )(A) and FRD2b (SEQ ID NO:13) (B) derived from the Ig1-to-lg2 reciprocal binding site of FGFR The length of neurites in cultures presented as a percentage of neurite length in the treated cultures compared to control (untreated cultures).
  • FGFR Fibroblast Growth Factor Receptor
  • Ig modules imunoglogulin-like modules
  • the invention relates to a functional cell-surface FGFR.
  • functional cell-surface receptor is meant a receptor that is located in the outer plasma membrane of the cell and has an identifiable group of extracellular ligands.
  • a "ligand” is any molecule that binds to a specific site in the receptor molecule. Binding of ligands to the recep- tor at specific sites usually occurs extracellularly and typically causes a change in the receptor molecule which is transferred further through the membrane and induces intracellular signal transduction which results in a physiological response of the cell.
  • the physiological response of a cell depends on which of the specific binding sites of a receptor is/are occupied by a ligand, on intracellular and extracellular environments of the receptor and/or on particularity of the ligand-receptor interaction, e.g. affinity and/or duration of interaction.
  • Ligand binding to a receptor often leads to a change in the activation status of the receptor, e.g. the receptor becomes capable of initiating a cascade of biochemical reactions inside the cell resulting in one or more of the above mentioned cellular responses. Binding of the ligand may also results in the inhibition of receptor activity which means that the receptor becomes unable of initiating a cascade of biochemi- cal reactions which is normally initiated due to ligand binding.
  • a group of FGFR ligands so far described in the art includes fibroblast growth factors (FGFs), heparin and neural cell adhesion molecules NCAM, L1 and N-cadherin. Binding sites for the latter ligands are different and located in the extracellular part of FGFR molecule.
  • FGFs fibroblast growth factors
  • NCAM neural cell adhesion molecules
  • L1 and N-cadherin binding sites for the latter ligands are different and located in the extracellular part of FGFR molecule.
  • One individual FGFR molecule can also extracellularly bind another individual FGFR molecule at a specific binding site located in the Ig2 module making thus a- FGFR dimer.
  • the FGFR-to-FGFR binding in essential for FGFR activity and occurs in the course of the ligand binding, being thus a ligand assisted - receptor binding/dimerization. Spontaneous dimerisation of two individual FGFR molecules takes place seldom and usually associated with pathological
  • an individual FGFR molecule comprising Ig1 and Ig2 modules separated by a linker region comprises a binding site for the recoprocal interaction if the Ig1 and Ig2 module interaction of which prevents interaction of said individual FGFR molecule wit another individual FGFR molecule and thus prevent dimerisation of FGFR.
  • the reciprocal interaction of Ig 1 and Ig2 modules involves specific amino acid residues of the modules.
  • the interacting amino acid residues of the reciprocal binding site include residues of both the Ig1 and Ig2 modules of the same FGFR molecule.
  • the reciprocal interaction of the Ig1 and Ig2 modules of the same FGFR polypeptide prevents interaction said FGFR polypeptide with another FGFR polypeptide and thus prevents FGFR self-activation at the absence of FGFs.
  • the reciprocal binding of the Ig 1 and Ig2 modules of the same FGFR mole- cule according to the invention also attenuates activating of FGFR by a ligand, e. g. a FGF, heparin, cell adhesion molecule, by decreasing the affinity of the FGFR - ligand interaction and/or decreasing the stability of the FGFR-ligand receptor complex, inhibiting thus FGFR activity.
  • the invention relates to a reciprocal Ig1-to-lg2 binding site in FGFR and to compounds that are capable of modulating the interaction of the Ig1 and Ig2 modules at this binding site.
  • the compounds of the invention are isolated peptides which comprise a fragment of FGFR comprising amino acid residues involved in the reciprocal interaction of the Ig1 and Ig2 modules. It is a preferred embodiment of the invention that the peptides comprise a fragment of FGFR which comprises a contiguous amino acid sequence derived from the reciprocal Ig1-to-lg2 binding site. In one embodiment, the peptide may comprise a contagious amino acid sequence which is derived from a part of the reciprocal Ig1-to-lg2 binding site which comprises amino acid residues of the Ig 1 module. In another embodiment, the peptide according to the invention may comprise a contiguous amino acid sequence which is derived from a part of the reciprocal Ig1-to-lg2 binding site which comprises amino acid residues of the Ig2 module.
  • the peptide according to invention may be capable of interacting with the Ig1 module of a FGFR of at the reciprocal Ig1-to-lg2 binding site.
  • the peptide according to the invention may be capable of interacting with the Ig2 module of a FGFR at the reciprocal Ig1-to-lg2 binding site.
  • a peptide of the invention is capable of binding to any FGFR, such as FGFR1 , FGFR2, FGFR3, FGFR4 and FGFR5 or it may bind to a variant of any of FGFR1-5, such as a natural or recombinant FGFR variant, for example a FGFR variant produced due alternative splicing, e.g. FGFRIb or FGFR2b, or genetic polymorphism, or any type of recombinant FGFR.
  • a FGFR of the invention and a variant thereof comprise the reciprocal Ig1-to-lg2 module binding site described herein, or comprise at least a part of said reciprocal binding site.
  • FGFRs of the invention which comprise the reciprocal binding site of the invention may be the FGFR polypeptides identified in the GenBank database as Ass. Nos: P11362 (corresponding to human FGFR1), P21802 (corresponding to human FGFR2), P22607 (corresponding to human FGFR3), P22455 (corresponding to human FGFR4) or AAK26742 (corresponding to human FGFR5).
  • the peptide according to the invention is a peptide which is capable of modulating activity of FGFR.
  • the peptide may be capable of activating FGFR.
  • the peptide may be capable of inhibiting FGFR.
  • FGFR of the invention is FGFR1 or a variant thereof.
  • FGFR1 may be activated by the peptide of the invention.
  • FGFR1 may be inhibited by the peptide of the invention.
  • Peptides according to the invention comprise a fragment of FGFR which comprises a contigous amino acid sequence derived from the reciprocal Ig1-to-lg2 binding site.
  • amino acid sequence according to the invention may be selected from the following amino acid sequences:
  • TKYQISQPEV SEQ ID NO:2
  • EPGQQEQLV SEQ ID NO:3
  • EASEEVELEPCLA SEQ ID NO: 7
  • EKMEKKLHAV SEQ ID NO:8
  • QRMEKKLHAV (SEQ ID NO:11 )
  • AANTVKFRC (SEQ ID NO:14)
  • AANTVRFRC (SEQ I D NO: 15)
  • AGNTVKFRC (SEQ ID NO:16) or
  • the peptide according to the invention may comprise a contigious amino acid sequence which is derived from a part of the reciprocal Ig1-to-lg2 binding site which comprises amino acid residues of the Ig1 module of FGFR. Accordingly, in this embodiment the invention concerns the following se- quences:
  • the amino acid sequences identified as SEQ ID NOs: 1-7 all comprise a binding motif which is essential for interaction of said sequences with amino acid residues of the Ig1-to-lg2 reciprocal binding site of FGFR located in the Ig2 module, said binding motif has the following formula Q/E-(x) 3 -x p , wherein x p is a hydrophobic amino acid residue and (x) 3 is an amino acid sequence of three amino acid resides, wherein x is any amino acid residue.
  • the residues x p may be any hydrophobic amino acid residue, however residues V, L or P are preferred.
  • amino acid sequence (x) 3 comprises at least one residue Q or at least one residue E, even more preferred that the (x) 3 sequence further comprises either a charged amino acid residue or a phydrophobic amino acid residue.
  • a preferred hydrophobic residue may be selected from P, I, L or V. Most prefered the motif which correspond to any the following amino acid sequences: QAQPW (SEQ ID NO: 18) QISQP (SEQ ID NO: 19)
  • the peptide according to the invention may com- prise a contigious amino acid sequence which is derived from a part of the reciprocal Ig1-to-lg2 binding site which comprises amino acid residues of the Ig2 module. Accordingly, in this embodiment the invention relates to the following sequences: EKMEKKLHAV (SEQ ID NO:8) EKMEKRLHAV (SEQ ID NO:9)
  • AAKTVKFKC (SEQ I D NO: 13)
  • AANTVKFRC (SEQ ID NO:14)
  • AANTVRFRC (SEQ ID NO:15)
  • VGSSVRLKC (SEQ ID NO:17).
  • sequences SEQ ID NOs:8-17 comprise a binding motif which is essential for interaction of said sequences with amino acid residues of the Ig1-to-lg2 reciprocal binding site of FGFR located in the Ig1 module.
  • the binding motif is defined by the formula K/R-x p -(x) Q-r K/R, wherein x p is a hydrophobic amino acid residue and (x) is a charged amino acid residue.
  • x p is any hydrophobic amino acid residue, however residues M, L or F in position x p are preferred.
  • the amino acid sequence comprises a three-amino-acid motif K/R-x p -(x) 0 -K/R , wherein x p is F or L, in another embodiments it may be preferred that the sequence comprises a four-amino-acid motif K/R-x p -(x) r K/R, wherein x p is M. Most prefered a motif which corresponds to an amino acid sequence selected from the following amino acid sequences:
  • RMDK (SEQ ID NO: 27)
  • RFR (SEQ ID NO: 32)
  • RLK (SEQ ID NO: 33).
  • the invention relates to SEQ ID NOs: 1-17 and SEQ ID NOs: 18-33 as preferred amino acid sequences comprised by the peptide of the invention.
  • the C-terminal amino acid of a peptide of the invention exists as the free carboxylic acid, this may also be specified as "-OH".
  • the C-terminal amino acid of a compound of the invention may be the amidated derivative, which is indicated as "-NH 2 ".
  • the N-terminal amino acid of a polypeptide comprise a free amino-group, this may also be specified as "H-”.
  • amino acid can be selected from any amino acid, whether naturally occurring or not, such as alfa amino acids, beta amino acids, and/or gamma amino acids. Accordingly, the group comprises but are not limited to: A, V, L, I 1 P 1 F, W, M, G, S 1 T, C, Y, N, Q, D, E 1 K, R 1 H Aib, NaI, Sar, Orn, Lysine analogues, DAP, DAPA and 4Hyp.
  • modifications of the amino acid sequences may be performed, such as for example glycosylation and/or acetylation of the amino acids.
  • Basic amino acid residues are according to invention represented by the residues of amino acids H, K and R; acidic amino acid residues - by the residues of amino acids E and D; hydrophobic amino acid residues by the residues of amino acids A, L, I, V 1 M, F, Y and W; neutral, weakly hydrophobic - by P, A and G; neutral hydrophilic - by amino acid residues Q, N, S and T; cross-link forming by amino acid resudue C.
  • a preferred peptide according to the invention is an isolated contigous peptide sequence which comprises at most 25 amino acid residues.
  • the length of the amino acid sequence of a peptide may be from 15 to 25 amino acid residues, such as, for example 16, 17, 18, 19, 20, 21, 22, 23 or 24 amino acid residues.
  • the length of the amino acid sequence of a peptide may be from 3 to 15 amino acid residues, such as for example 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid residues.
  • the peptides which amino acid sequence has the length in the range of 5 to 15 amino acid residues, such as from 6 to 14, for example 7, 8, 9, 10 ,11 12 or 13, are preferred. It is understood that all peptides of the invention comprise at least one amino acid sequence selected from any of the sequences SEQ ID NOs: 1-17 or at least one fragment of any of these sequences.
  • the invention may relate to a peptide comprising a fragment of an sequence selected from SEQ ID NOs:1-17.ln another embodiments, the invention may relates to variants of SEQ ID NOs:1-17.
  • a variant of an amino acid sequence selected from the sequences SEQ ID NOs: 1-17 may be i) an amino acid sequence which has at least 60% identity with a selected sequence, such as 61-65% identity, for example 66-70% identity, such as 71-75% identity, for example 76-80% identity, such as 81-85 % identity, for example 86-90% identity, such as 91-95% identity, for example 96- 99% identity, wherein the identity is defined as a percentage of identical amino acids in said sequence when it is collated with the selected sequence.
  • the identity between amino acid sequences may be calculated using well known algorithms such as BLOSUM 30, BLOSUM 40, BLO-
  • an amino acid sequence which has at least 60% positive amino acid matches with a selected sequence such as 61-65% positive amino acid matches, for example 66-70% positive amino acid matches, such as 71- 75% positive amino acid matches, for example 76-80% positive amino acid matches, such as 81-85 % positive amino acid matches, for example 86-90% positive amino acid matches, such as 91-95% positive amino acid matches, for example 96-99% positive amino acid matches, wherein the positive amino acid match is defined as the presence at the same position in two compared sequences of amino acid residues which has similar of physical and/or chemical properties.
  • Preferred positive amino acid matches of the present invention are K to R, E to D, L to M, Q to E, I to V,
  • an amino acid sequence which is identical to a selected sequence, or it ⁇ has at least 60% identity with said sequence such as 61-65% identity, for example 66-70% identity, such as 71-75% identity, for example 76-80% identity, such as 81-85 % identity, for example 86-90% identity, such as 91-95% identity, for example 96-99% identity, or has at least 60% positive amino acid matches with the selected sequence, such as 61-65% positive amino acid matches, for example 66-70% positive amino acid matches, such as 71-75% positive amino acid matches, for example 76-
  • 80% positive amino acid matches such as 81-85 % positive amino acid matches, for example 86-90% positive amino acid matches, such as 91- 95% positive amino acid matches, for example 96-99% positive amino acid matches and comprises other chemical moieties, e. g. phosphoryl, sulphur, acetyl, glycosyl moieties.
  • variant of a peptide sequence also means that the peptide sequence may be modified, for example by substitution of one or more of the amino acid residues. Both L-amino acids and D-amino acids may be used. Other modification may comprise derivatives such as esters, sugars, etc., for example methyl and acetyl esters.
  • variants of the amino acid sequences according to the invention may comprise, within the same variant, or fragments thereof or among different variants, or fragments thereof, at least one substitution, such as a plurality of substitutions introduced independently of one another.
  • Variants of the complex, or fragments thereof may thus comprise conservative substitutions independently of one another, wherein at least one glycine (GIy) of said variant, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Ala, VaI, Leu, and lie, and independently thereof, variants, or fragments thereof, wherein at least one alanine (Ala) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of GIy, VaI, Leu, and lie, and independently thereof, variants, or fragments thereof, wherein at least one valine (VaI) of said variant, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of GIy, Ala, Leu, and lie
  • the groups of conservative amino acids are as the following: A, G (neutral, weakly hydrophobic), Q, N, S, T (hydrophilic, non-charged) E, D (hydrophilic, acidic) H, K, R (hydrophilic, basic) L, P, I, V, M, F, Y, W (hydrophobic, aromatic) C (cross-link forming) Conservative substitutions may be introduced in any position of a preferred predetermined peptide of the invention or fragment thereof. It may however also be desirable to introduce non-conservative substitutions, particularly, but not limited to, a non-conservative substitution in any one or more positions.
  • a non-conservative substitution leading to the formation of a functionally equivalent fragment of the peptide of the invention would for example differ substantially in polarity, for example a residue with a non-polar side chain (Ala, Leu, Pro, Trp, VaI, He, Leu, Phe or Met) substituted for a residue with a polar side chain such as GIy, Ser, Thr, Cys, Tyr, Asn, or GIn or a charged amino acid such as Asp, GIu, Arg, or Lys, or substituting a charged or a polar residue for a non-polar one; and/or ii) differ substantially in its effect on peptide backbone orientation such as substitution of or for Pro or GIy by another residue; and/or iii) differ substantially in electric charge, for example substitution of a negatively charged residue such as GIu or Asp for a posi- tively charged residue such as Lys, His or Arg (and vice versa); and/or iv) differ substantially in ster
  • Substitution of amino acids may in one embodiment be made based upon their hy- drophobicity and hydrophilicity values and the relative similarity of the amino acid side-chain substituents, including charge, size, and the like
  • a fragment of a selected amino acid sequence of the invention may be an amino acid sequence, which has about 25 - 99 % of the length of the selected amino acid sequence.
  • a fragment according to the invention comprises at least 3 contigous amino acid residues of any of the seaquences SEQ ID NOs:1-17, such as for example a sequence selected from SEQ ID NOs:18-33.
  • the term “functional equivalent" of an amino acid sequence is in the present context meant a molecule which meets the criteria for a variant or a fragment of said amino acid sequence described above and which is capable of one or more func- tional activities of said sequence or a compound comprising said sequence, in a preferred embodiment the functional equivalent of an amino acid sequence of the invention is capable of binding and modulating activity of a FGFR.
  • the invention relates both to isolated peptides of the invention and fusion proteins comprising peptides of the invention.
  • the peptide is an isolated peptide.
  • isolated peptide is meant that the peptide of the invention is an individual compound and not a part of another compound, such as for example a polypeptide comprising more then 25 amino acid residues.
  • the isolated peptide may be produced by use of any recombinant technology methods or chemical synthesis and separated from other compounds, or it was separated from a longer polypeptide or protein by a method of enzymatic or chemical cleavage and further separated from other protein fragments.
  • An isolated peptide of the invention may in one embodiment comprise one or more of the sequences SEQ ID NOs: 1-33. In another embodiment the isolated peptided may consist of one or more of the sequences SEQ ID Nos:1-33.
  • the peptide comprises a sequence selected from SEQ ID NOs: 1-8 or 18-28, preferably a sequence selected from SEQ ID NOs: 1-8, or a functional equivalent thereof.
  • the peptide may consist of a sequence selected from SEQ ID NOs: 1-8 or a functional equivalent thereof.
  • the peptide may comprise a sequence selected from SEQ ID NOs: 9-17 or 26-33, preferably a sequence selected from SEQ ID NOs: 9-17, or a functional homologue thereof.
  • a preferred amino acid sequence may be chosen depending on which type of modu- lating FGFR activity is nessesary. Thus, in one embodiment it may be nessesary to activate FGFR, in another embodiment it may be nessesary to attenuate activity of
  • FGFR or inhibit the receptor.
  • the chose of a peferred amino acid se- quennce sequence may depend on which receptor of the FGFRs is concerned.
  • FGFR1 may be concerned, in another it may be con- cerned FGFR2 or FGFR3.
  • it may be nessesary to modu- late activity of FGFR4.
  • the choice of a preferred amino acid sequence depending on which of the FGFRs is concerned is sometimes of less importance, as the amino acid sequences described above according to the invention are capable of interacting with the reciprocal Ig1-to-lg2 module binding site of any FGFR.
  • An isolated peptide sequence of the invention may be connected to another isolated peptide sequence by a chemical bond in a fusion protein or the amino acid se- quences may be connected to each other through a linker grouping.
  • a peptide sequence of the invention may be formulated as an oligomer (multimer) of monomers, wherein each monomer is as a peptide seqience defined above.
  • multimeric peptides such as dendrimers may form conformational determinants or clusters due to the presence of multiple flexible peptide monomers.
  • the compound is a dimer.
  • the compound is a dendrimer, such as four peptides linked to a lysine backbone, or coupled to a polymer carrier, for example a protein carrier, such as BSA.
  • a polymer carrier for example a protein carrier, such as BSA.
  • Polymerisation such as repetitive sequences or attachment to various carriers are well-known in the art, e.g. lysine backbones, such as lysine dendrimers carrying 4 peptides, 8 peptides, 16 peptides, or 32 peptides.
  • Other carriers may be lipophilic dendrimers, or micelle-like carriers formed by lipophilic derivatives, or starburst (star-like) carbon chain polymer conjugates.
  • a multimeric compound may be a polymer compris- ing two or more identical or different peptide sequences of the invention, wherein in a preferred embodiment, at least one of the two or more amino acid sequences is selected from SEQ ID NOs: 1-15, or fragments or variants of said sequences.
  • the compound may comprise two identical amino acid se- quences selected from SEQ ID NOs: 1-15, or two identical fragments or variants of the selected sequence, wherein said amino acid sequences, fragments or variants.
  • the compound may comprise four identical copies of an amino acid sequence selected from SEQ ID NOs: 1-15, or four identical fragments or variants of the selected sequence.
  • the compound may comprise two or more different amino acid sequences, wherein at least one of the two amino acid sequences is a sequence selected from SEQ ID NOs: 1-15, or fragments or variants thereof.
  • the compound may comprise two or more different amino acid sequences, wherein said two or more amino acid sequences are selected from SEQ ID NO: 1-15, or fragments or variants thereof.
  • a preferred multimeric compound of the invention is a compound wherein the amino acid sequences are connected to each other through a linker or a linker grouping.
  • a linker is according to the invention may be any molecule or chemical moiety capable of cross-linking two or more peptide sequences, for example it may be an achiral di-, tri- or tetracarboxylic acid of the general formula
  • n and m independently are an integer of from 1 to 20,
  • X is HN, H 2 N(CR 2 )PCR, RHN(CR 2 )pCR, HO(CR 2 )pCR, HS(CR 2 )pCR, halogen- (CR 2 )PCR, HOOC(CR 2 )pCR, ROOC(CR 2 )pCR, HCO(CR 2 )pCR, RCO(CR 2 )pCR, [HOOC(A)n][HOOC(B)m]CR(CR 2 )pCR, H 2 N(CR 2 )P, RHN(CR 2 )p, HO(CR 2 )p, HS(CR 2 )P, halogen-(CR 2 )p, HOOC(CR 2 )p, ROOC(CR 2 )P, HCO(CR 2 )P, RCO(CR 2 )p, or [HOOC(A)n][HOOC(B)m](CR 2 )p , wherein
  • C 1-10 alkyl straight or branched chain alkyl groups having 1- 10 carbon atoms, e.g. methyl, ethyl, isopropyl, butyl, and tertbutyl.
  • C 2-10 alkenyl is meant straight or branched chain alkenyl groups hav- ing 2-10 carbon atoms, e.g. ethynyl, propenyl, isopropenyl, butenyl, and tert-butenyl.
  • cyclic moiety is meant cyclohexan, and cyclopentane.
  • aromatic moiety is meant phenyl
  • a and B forms a cyclic, heterocyclic or aromatic moiety denotes cyclohexan, piperidine, benzene, and pyridine.
  • a multimeric compound of the invention comprises the linker of above
  • the compound is preferably obtained by the LPA method (a ligand presentation assembly method) as described in WO0018791 and WO2005014623.
  • a preferred linker of the invention may be amino acid lysine.
  • Individual peptide sequences may be attached to a core molecule such as lysine forming thereby a dendritic multimer (dendrimer) of an individual peptide sequence(s).
  • dendrimers Production of dendrimers is also well known in the art (PCT/US90/02039, Lu et al., (1991) MoI Immunol. 28:623-630; Defoort et al., (1992) lnt J Pept Prot Res. 40:214-221 ; Drijfhout et al. (1991) lnt J Pept Prot Res. 37:27- 32), and dedrimers are at present widely used in research and in medical applications.
  • amino acid cystein may be preferred a linker molecule.
  • One of the referred embodiments of the invention concernes a compound comprising four individual amino acid sequences attached to the lysine core molecule, a dendritic tetramer/dendrimer of a peptide sequence of the invention.
  • Multimeric compounds of the invention are most preferred compounds of the invention.
  • other types of multimeric compounds comprising two or more individual sequences of the invention are also in the scope of the invention. These compounds may be produced using thechnologies known in the art.
  • the peptide sequences may be covalently bound to the linker through their amino- or carboxy-groups, preferably through the N- or C terminal amino- or carboxy- groups.
  • compounds described above are functionally active compounds.
  • the term "compound” relates in the present context both to isolated peptide sequences of the invention and compounds comprising said sequences.
  • the compounds are capable of binding to a functional cell-surface receptor and modulating the activity of said receptor
  • the receptor is according to the invention is FGFR and may be selected from any of the FGFRs, for example it may be FGFR1, FGFR2, FGFR3, FGFR4 or FGFR5.
  • the compound according to the invention is capable of binding to any of the latter FGFRs at the reciprocal Ig1-to-lg2 binding site located in the Ig1 and Ig2 modules of FGFR.
  • the invention preferably concerns FGFR1 and FG FR1 -associated signalling.
  • the modulating FGFR1 signalling by the compound of the invention results in a change in the receptor activation status which is reflected by FGFR1 tyrosine phosphorylation, or it may be reflected by the status of activation of one or more of the intracellular proteins involved in FGFR1 -associated signal transduction, such as for example the activation status of STAT1 , JNK, PI_C ⁇ , ERK, STAT5, PI3K, PKC, FRS2 and/or GRB2 proteins.
  • the result of modulating of FGFR1 signalling by a compound of the invention may also be related to a cell differentiation-related effect.
  • the degree of phoshorylation is estimated as at least 20% above the control value, such as at least 20-200 %, for example at least 50-200%.
  • the control value in the present content is meant the degree of phosphorylation of FGFR in the medium where a compound capable of activation of FGFR is absent.
  • said concentration may be between 0.1-1000 ⁇ M, 1- 1000 ⁇ M, for example 1-200 ⁇ M, for example 10-200 ⁇ M, such as 20-180 ⁇ M, for example 30-160 ⁇ M, such as 40-140 ⁇ M, for example 50-130 ⁇ M, such as 60-120 ⁇ M, for example 70-110 ⁇ M, such as 80-100 ⁇ M.
  • the invention preferably relates to cell aggregation, the formation of nodules, formation of cartilage, or two or more of said effects (Listrum, G. P. et al. J. Histochem. Cytochem. 1999, 47:1-6), such effect being detectable by light microscopy, turbidimetry, or flow cytometry.
  • the cell differentiation-related effect may also be measured as a change in expression at RNA or protein levels of bone sialo- protein (J. Bone Miner. Res. 1998, 13:1852-61; Genomics 1998, 53: 391-4), or type X collagen (Cell Tissue Res. 1998, 293: 357-64), the human ILA gene (Osteoarthritis Gartilage 1997, 5: 394-406), or type Il collagen/or MGP (J Miner Res. 1997: 1815.23), and the like.
  • the FGFR tyrosine phosphorylation or activation of any of the molecules of FGFR- associated downstream signaling may be estimated by any conventional methods, such as for example immunocytochemistry, immunoblotting or im- munoprecipitation, using commercially available antibody against the activated pro- teins.
  • the degree of activation is estimated as at least 20% above/below the control value, such as at least 20-200 %, for example at least 50-200%.
  • the control value is estimated as a degree of phosphorylation of the protein of interest in the medium where a compound capable of activation of FGFR is absent.
  • the invention relates to a signaling associated with a protein involved in interaction with FGFR, for example the protein being an FGFR1 ligand, preferably a receptor-like ligand FGFR1.
  • FGFR1 FGFR1
  • FGFR1 FGFR1
  • receptor-like ligand FGFR1 are neural cell adhesion molecules NCAM and L1. It is understood that receptor-like FGFR ligands may comprise other proteins, which are capable of inter- acting with FGFR and associated with any signal transduction cascade.
  • the most preferred a receptor-like ligand of FGFR1 of the invention is NCAM. Therefore, the invention concerns biological effects which are associated with FGFR-NCAM interaction, for example such as neurite outgrowth or neural cell dif- ferentiation.
  • NCAM-dependent signal transduction involves the variety of downstream molecules, activation of which upon the signalling may be measured.
  • the invention in particular concerns the assessment of activation of focal adhesion kinase FAK, tyrosine kinase Fyn and/or cyclic-AMP response-binding element protein CREB.
  • the degree of phoshorylation is estimated as at least 20% above/below the control value, such as at least 20-200 %, for example at least 50-200%.
  • the control value is estimated as above.
  • Activation or inhibition of NCAM-dependent signal transduction may also be measured by evaluating the cellular responses on morphological level, in particular cell differentiation-related effects. Accordingly, the assay concerns in another particular embodiment evaluation of the effect of a candidate compound on NCAM-dependent cellular aggregation, cell motility, neuritogenesis, survival, plasticity associated with memory and learning.
  • Cellular aggregation and neuritogenesis may for example be evaluated as described by Skladchikova et al. J. Neurosci. Res 1999, 57: 207-18.
  • Proliferation and apoptosis may be evaluated by using any commercially available assays and kits according to the manufacturer procedure.
  • compounds of the invention are capable of activating FGFR directly by binding to the reciprocal binding site described herein, or they may attenuate FGFR acti- vating dependent on other ligands.
  • the compounds of the invention may modulate receptor signalling dependent on another ligand binding.
  • a cellular response to the activation of a receptor depends on the strength of receptor stimulation, which may, for example, be characterised by the value of affinity of interaction of a ligand with the receptor, and/or by the duration of such interaction.
  • affinity and duration of interaction of FGF and the receptor may be affected by a compound of the invention.
  • interacting is used interchangeably with the term “binding” and refers to a direct or indirect contact between a compound of the invention and FGFR, preferably a direct interaction.
  • direct interaction means that the compound in question binds directly to the receptor.
  • the binding affinity of the compound according to the invention preferably has Kd value in the range of 10 "3 to 10 "10 M, such as preferably in the range of 10 "4 to 10 "8 M.
  • the binding affinity may be determined by any available assayes suitable for this purpose, such as for example surface plasmon resonance (SPR) analysis or nuclear magnetic resonance (NMR) spectroscopy.
  • Binding of the compound of the invention to FGFR leads to a series of cellular re- sponces mediated by FGFR.
  • the compound which is capable of binding to FGFR and activating/inhibiting FGFR is also capable of inducing differentiation of FGFR presenting cells, modulating of proliferation of FGFR presenting cells, stimulating survival of FGFR presenting cells, and /or stimulating morphological plasticity of FGFR presenting cells.
  • cells presenting FGFR cells expressing FGFR on the external membrane of the cells, these cells are for example neurons, glial cells, all types of muscle cells, neuroendocrine cells, gonadal cells and kidney cells, endothelial cells fibroblasts, osteoblasts, cancer, stem and embryonic cells. Activation of FGFRs has been shown to be associated with growth, differentiation and survival of cells ex- pressing the receptors.
  • FGFRs have been shown to be important determinants of neuronal survival both during development and during adulthood (Haspel et al. (2000) J Neurobiol 15:287-302; Roonprapurt et al. (2003) J Neurotrauma 20:871-882; Wiencken-Barger et al. Cereb Cortex (2004) 14:121-131; Loers er al. (2005) J Neurochem 92:1463- 1476; Reuss and von Bohlen und Halbach (2003) Cell tissue Res, 313:139-57), muscle and cancer cells (Ozen et al. (2001) J Nat Cancer Inst. 93:1783-90; Miyamoto et al. (1998) J Cell Physiol.
  • FGF receptors Activation of FGF receptors is involved in normal, as well as in pathologic angiogenesis (Slavin, Cell Biol lnt 1995, 19:431-44). It is important for development, proliferation, functioning and survival skeletal muscle cells, cardiomyocytes and neurons (Merle at al., J Biol Chem 1995, 270:17361-7; Cheng and Mattson, Neuron 1991, 7:1031-41; Zhu et al., Mech Ageing Dev 1999, 108:77-85). Alterations in FGFR signalling have been associated with development different pathologic condi- tions, e.g.
  • substances with the potential to stimulate FGFR signalling have the potential to promote neurite outgrowth as well as stimulate regeneration and/or differentiation of neuronal cells, to stimulate cell survival, in particular neuronal cell survival, to stimulate stem or premature cell differentiation, such as neuronal cell differentiation, to stimulate neuronal plasticity associated with memory and learning, are prime targets in the search for compounds that facilitate for example neuronal regeneration and other forms of neuronal plasticity.
  • Compounds of the present invention are shown to promote neurite outgrowth and are therefore considered to be good promoters of regeneration of neuronal connec- tions, and thereby of functional recovery after damages as well as promoters of neuronal function in other conditions where such effect is required. Furthermore, compounds of the present invention are capable of stimulating neuronal progenitor cell differention into marture neurons. Compounds of the present invention are also potent stimulators of morphological plasticity of neurons associated with learning and memory.
  • differentiation is related both to the processes of initiation of differentiation of neuronal precursor cells/stem, maturation of immature neurons, such as neurite outgrowth which take place after the last cell division of said neurons, and morphological plasticity of mature neurons, such as takes place in the brain in connection with learning and memory.
  • the compounds of the present invention may be capable of stopping neural precursor and immature neural cell division and initiating maturation said cells, such as initiating extension of neurites.
  • differentiation is related to initiation of the process of genetic, biochemical, morphological and physiological transformation of neuronal progenitor cells, immature neural cells or embryonic stem cells leading to formation of cells having functional characteristics of normal neuronal cell as such characteristics are defined in the art.
  • the invention defines "immature neural cell” as a cell that has at least one feature of neural cell accepted in the art as a feature characteristic for the neural cell.
  • a compound comprising at least one of the above peptide sequences is capable of stimulating neurite outgrowth.
  • the invention concerns the neurite outgrowth improvement/stimulation such as about 75% improvement/stimulation above the value of neurite outgrowth of control/non- stimulated cells, for example 50%, such as about 150%, for example 100%, such as about 250, for example 200%, such as about 350 %, for example 300%, such as about 450%, for example 400%, such as about 500%.
  • Estimation of capability of a candidate compound to stimulate neurite outgrowth may be done by using any known method or assay for estimation of neurite outgrowth, such as for example as the described in Examples.
  • a compound has neuritogenic activity both as an insoluble immobile component of cell growth substrate and as a soluble component of cell growth media.
  • immobile means that the compound is bound/attached to a substance which is insoluble in water or a water solution and thereby it becomes insoluble in such solution as well.
  • insoluble and soluble compounds are considered by the application, however soluble compounds are preferred.
  • soluble compound is understood a compound, which is soluble in water or a water solution.
  • the compound of the invention is capable of stimulating synaptic plasticity. Accordingly, the compound is capable of stimulating empering and memory as well.
  • the peptide sequences of the invention may stimulate spine formation, in another embodiment the sequences may promote synaptic efficacy.
  • the invention further provides a method for stimulating memory and/or learning comprising using a peptide sequence of the invention and/or compound comprising said sequence. The invention relates to both short-term memory and long-term memory.
  • the compound of the invention is capable of stimulating cell survival.
  • the compounds according to the invention are capable to prevent of cell death, in particular neuronal cell death, for example cell death due to trauma or disease.
  • the wording "stimulate/promote survival” is used synonymously with the wording "preventing cell death”.
  • stimulating/promoting cell survival it is possible to prevent diseases or prevent further degeneration of the nervous system in individuals suffering from a degenerative disorder.
  • “Survival” refers to the process, wherein a cell has been traumatised and would under normal circumstances, with a high probability die, if not a compound of the invention was used to prevent said cell from degenerating, and thus promoting or stimulating survival of said traumatised cell.
  • Peripheral nerve cells possess to a limited extent a potential to regenerate and re- establish functional connections with their targets after various injuries. However, functional recovery is rarely complete and peripheral nerve cell damage remains a considerable problem. In the central nervous system, the potential for regeneration is even more limited. Therefore, the identification of substances with the ability to prevent neuronal cell death in the peripheral and the central nervous system is of great interest.
  • the invention also relates to compounds that are capable of attenuating FGFR function, such as inhibiting FGFR activity.
  • Pathologic activity of FGFRs have been shown associated with distinct clinical entities, including achondroplasia, hypochondroplasia, platyspondylic lethal skeletal dysplasia, thanatophoric dysplasia, Antley-Bixler syndrome, Apert syndrome, Beare-Stevenson syndrome, Crouzon syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, and Saethre-Chotzen syndrome (Passos-Bueno et al., 1999H ⁇ /m. Mutat. 14: 115-125).
  • the compound described above is ca- pable of inhibiting FGFR.
  • the peptide sequences of the present invention may be prepared by any conven- tional synthetic methods, recombinant DNA technologies, enzymatic cleavage of full-length proteins which the peptide sequences are derived from, or a combination of said methods.
  • the peptides of the invention are produced by use of recombinant DNA technologies.
  • the DNA sequence encoding a peptide or the corresponding full-length protein the peptide originates from may be prepared synthetically by established standard methods, e.g. the phosphoamidine method described by Beaucage and Caruthers, 1981 , Tetrahedron Lett. 22:1859-1869, or the method described by Matthes et al., 1984, EMBO J. 3:801-805.
  • oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in suitable vectors.
  • the DNA sequence encoding a peptide may also be prepared by fragmentation of the DNA sequences encoding the corresponding full-length protein of peptide origin, using DNAase I according to a standard protocol (Sambrook et al., Molecular clon- ing: A Laboratory manual. 2 rd ed., CSHL Press, Cold Spring Harbor, NY, 1989).
  • the present invention relates to full-length proteins selected from the groups of proteins identified above.
  • the DNA encoding the full-length proteins of the invention may alternatively be fragmented using specific restriction endonucleases.
  • the fragments of DNA are further purified using standard procedures described in Sambrook et al., Molecular cloning: A Laboratory manual.
  • the DNA sequence encoding a full-length protein may also be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the full-length protein by hybridisation using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989).
  • the DNA sequence may also be prepared by polymerase chain reaction using specific primers, for instance as described in US 4,683,202 or Saiki et al., 1988, Science 239:487-491.
  • a recombinant expression vector which may be any vector, which may conveniently be subjected to recombinant DNA procedures.
  • the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vec- tor that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the DNA sequence encoding a peptide or a full-length protein should be operably connected to a suitable promoter sequence.
  • the promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • suitable promoters for directing the transcription of the coding DNA sequence in mammalian cells are the SV 40 promoter (Subramani et al., 1981, MoI. Cell Biol. 1:854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al., 1983, Science 222: 809-814) or the adenovirus 2 major late promoter.
  • a suitable promoter for use in insect cells is the polyhedrin promoter (Vasu- vedan et al., 1992, FEBS Lett. 311:7-11).
  • Suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., 1980, J. Biol. Chem. 255:12073-12080; Alber and Kawasaki, 1982, J. MoI. Appl. Gen.
  • Suitable promoters for use in filamentous fungus host cells are, for in- stance, the ADH3 promoter (McKnight et al., 1985, EMBO J. 4:2093-2099) or the tpiA promoter.
  • the coding DNA sequence may also be operably connected to a suitable terminator, such as the human growth hormone terminator (Palmiter et al., op. cit.) or (for fungal hosts) the TPH (Alber and Kawasaki, op. cit.) or ADH3 (McKnight et al., op. cit.) promoters.
  • the vector may further comprise elements such as polyadenylation signals (e.g. from SV 40 or the adenovirus 5 EIb region), transcriptional enhancer sequences (e.g. the SV 40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs).
  • the recombinant expression vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • a DNA sequence enabling the vector to replicate in the host cell in question.
  • An example of such a sequence is the SV 40 origin of replication.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or one which confers resistance to a drug, e.g. neomycin, hydromycin or methotrexate.
  • DHFR dihydrofolate reductase
  • the coding DNA sequences may be usefully fused with a second peptide coding sequence and a protease cleavage site coding sequence, giving a DNA construct encoding the fusion protein, wherein the protease cleavage site coding sequence positioned between the HBP fragment and second peptide coding DNA, inserted into a recombinant expression vector, and expressed in recombinant host cells.
  • said second peptide selected from, but not limited by the group comprising glutathion-S-reductase, calf thymosin, bacterial thioredoxin or human ubiquitin natural or synthetic variants, or peptides thereof.
  • a peptide sequence comprising a protease cleavage site may be the Factor Xa, with the amino acid sequence IEGR, en- terokinase, with the amino acid sequence DDDDK,- thrombin, with the amino acid sequence LVPR/GS, or Acharombacter lyticus, with the amino acid sequence XKX, cleavage site.
  • the host cell into which the expression vector is introduced may be any cell which is capable of expression of the peptides or full-length proteins, and is preferably a eu- karyotic cell, such as invertebrate (insect) cells or vertebrate cells, e.g. Xenopus laevis oocytes or mammalian cells, in particular insect and mammalian cells.
  • suitable mammalian cell lines are the HEK293 (ATCC CRL-1573), COS (ATCC CRL-1650), BHK (ATCC CRL-1632, ATCC CCL-10) or CHO (ATCC CCL- 61) cell lines.
  • fungal cells may be used as host cells.
  • suitable yeast cells include cells of Saccharomyces spp. or Schizosaccharo- myces spp., in particular strains of Saccharomyces cerevisiae.
  • Other fungal cells are cells of filamentous fungi, e.g. Aspergillus spp. or Neurospora spp., in particular strains of Aspergillus oryzae or Aspergillus niger.
  • Aspergillus spp. for the expression of proteins is described in, e.g., EP 238 023.
  • the medium used to culture the cells may be any conventional medium suitable for growing mammalian cells, such as a serum-containing or serum-free medium containing appropriate supplements, or a suitable medium for growing insect, yeast or fungal cells. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type CuI- ture Collection).
  • the peptides or full-length proteins recombinantly produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g.
  • HPLC ion exchange chromatography, affinity chromatography, or the like.
  • Peptides may for example be synthesised by using Fmoc chemistry and with Acm- protected cysteins. After purification by reversed phase HPLC, peptides may be further processed to obtain for example cyclic or C- or N-terminal modified isoforms.
  • the methods for cyclization and terminal modification are well-known in the art and described in detail in the above-cited manuals.
  • the peptide sequences of the invention are produced synthetically, in particular, by the Sequence Assisted Peptide Synthesis (SAPS) method.
  • SAPS Sequence Assisted Peptide Synthesis
  • Peptides may be synthesised either batchwise in a polyethylene vessel equipped with a polypropylene filter for filtration or in the continuous-flow version of the polyamide solid-phase method (Dryland, A. and Sheppard, R.C., (1986) J.Chem. Soc. Perkin Trans. I 1 125 - 137.) on a fully automated peptide synthesiser using 9- fluorenylmethyloxycarbonyl (Fmoc) or tert. -Butyloxycarbonyl, (Boc) as N-a-amino protecting group and suitable common protection groups for side-chain functionality's.
  • FGFRs are known to be involved in a number of body processes in normal conditions and in disease, in particular in the neural system. These proceses include dif- ferentiation, proliferation, survival, plasticity and motility of cells.
  • Cell death plays a key role in normal neuronal development, where 50% of the developing neurons are eliminated through programmed cell death, and in the pathophysiology of neurodegenerative conditions, such as Alzheimer's and Parkinson's diseases.
  • FGFRs have been shown to be important determinants of neuronal sur- vival both during development and during adulthood (Haspel et al. (2000) J Neurobiol 15:287-302; Roonprapurt et al.
  • the invention features compounds that promote survival of neural cells and can be used as medicaments for the treatment of conditions involving neural cell death.
  • a compound of the invention may also be used as a medicament for promotion of survival of another type of cells, e.g.
  • the present invention provides compounds capable of modulating the activity of FGFRs. Consequently, said compounds are concerned by the invention as medicament for the treatment of diseases, wherein modulation of FGFR activity may be concsemed as essential for curing.
  • the medicament of the invention is in one embodiment for prevention and/or treatment of
  • diseases or conditions of the central and peripheral nervous system such as postoperative nerve damage, traumatic nerve damage, impaired myelination of nerve fibers, postischaemic damage, e.g. resulting from a stroke, Parkinson's disease, Alzheimer's disease, Huntington's disease, dementias such as multiin- farct dementia, sclerosis, nerve degeneration associated with diabetes mellitus, disorders affecting the circadian clock or neuro-muscular transmission, and schizophrenia, mood disorders, such as manic depression;
  • diseases or conditions of the muscles including conditions with impaired function of neuro-muscular connections, such as after organ transplan- tation, or such as genetic or traumatic atrophic muscle disorders; or for treatment of diseases or conditions of various organs, such as degenerative conditions of the gonads, of the pancreas such as diabetes mellitus type I and II, of the kidney such as nephrosis and of the heart, liver and bowel, and/or
  • cancer disease and/or 5) prion diseases.
  • the invention concerns cancer being any type of solid tumors requiring neoangio- genesis.
  • the invention concerns prion diseases selected from the group consisting of scrapie, Creutzfeldt-Jakob disease. It has been shown that FGFRs plays a distinct role in prion diseases (Castelnau et al. (1994) Exp Neurobiol. 130:407-10; Ye and Carp (2002) J MoI Neurosci. 18:179-88).
  • a compound of the invention is for the manufacture of a medicament for 1) promotion of wound-healing, and/or
  • the invention concerns the medicament for treating normal, degenerated or damaged FGFR presenting cells or cells presenting an FGFR ligand.
  • cells presenting an FGFR ligand is meant cells expressing a receptor or ligand whereto FGFR and/or parts of FGFR may bind (i.e. so-called counter receptor).
  • FGFR ligands are FGFs (fibroblast growth factors), NCAM, L1 or proteoglycans, such as heparin, heparan sulphateproteoglycans, and chondroitin sulphatepro- " ' teoglycans.
  • the medicament of the invention comprises an effective amount of one or more compounds as defined above, or a pharmaceutical composition comprising one or more compounds and pharmaceutically acceptable additives.
  • the invention in another aspect also concerns a pharmaceutical composition
  • a pharmaceutical composition comprising at least one compound of the invention.
  • a further aspect of the invention is a process of producing a pharmaceutical composition, comprising mixing an effective amount of one or more of the compounds of the invention, or a pharmaceutical composition according to the invention with one or more pharmaceutically acceptable additives or carriers.
  • the compounds are used in combination with a prosthetic device, wherein the device is a prosthetic nerve guide.
  • the present invention relates to a prosthetic nerve guide, characterised in that it comprises one or more of the compounds or the pharmaceutical composition as defined above. Nerve guides are known in the art.
  • the invention relates to use of a medicament and/or pharmaceutical composition comprising the compound of invention for the treatment or prophylaxis of any of the diseases and conditions mentioned below.
  • Such medicament and/or pharmaceutical composition may suitably be formulated for oral, percutaneous, intramuscular, intravenous, intracranial, intrathecal, in- tracerebroventricular, intranasal or pulmonal administration.
  • Injectables are usually prepared either as liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid prior to injection.
  • the preparation may also be emulsified.
  • the active ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof.
  • excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof.
  • the preparation may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH- buffering agents, or which enhance the effectiveness or transportation of the preparation.
  • Formulations of the compounds of the invention can be prepared by techniques known to the person skilled in the art.
  • the formulations may contain pharmaceutically acceptable carriers and excipients including microspheres, liposomes, microcapsules, nanoparticles or the like.
  • the preparation may suitably be administered by injection, optionally at the site, where the active ingredient is to exert its effect.
  • Additional formulations which are suitable for other modes of administration include suppositories, nasal, pulmonal and, in some cases, oral formulations.
  • traditional binders and carriers include polyalkylene glycols or triglycerides.
  • Such suppositories may be formed from mixtures containing the active ingredient(s) in the range of from 0.5% to 10%, preferably 1-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sus- tained release formulations or powders and generally contain 10-95% of the active ingredient(s), preferably 25-70%.
  • formulations are such suitable for nasal and pulmonal administration, e.g. inhalators and aerosols.
  • the active compound may be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide compound) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with the free carboxyl group may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as iso- propylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the preparations are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective.
  • the quantity to be administered depends on the subject to be treated, including, e.g. the weight and age of the subject, the disease to be treated and the stage of disease. Suitable dosage ranges are per kilo body weight normally of the order of several hundred ⁇ g active ingredient per administration with a preferred range of from about 0.1 ⁇ g to 5000 ⁇ g per kilo body weight.
  • the suitable dosages are often in the range of from 0.1 ⁇ g to 5000 ⁇ g per kilo body weight, such as » in the range of from about 0.1 ⁇ g to 3000 ⁇ g per kilo body weight, and especially in the range of from about 0.1 ⁇ g to 1000 ⁇ g per kilo body weight.
  • the suitable dosages are often in the range of from 0.1 ⁇ g to 1000 ⁇ g per kilo body weight, such as in the range of from about 0.1 ⁇ g to 750 ⁇ g per kilo body weight, and especially in the range of from about 0.1 ⁇ g to 500 ⁇ g per kilo body weight such as in the range of from about 0.1 ⁇ g to 250 ⁇ g per kilo body weight.
  • Administration may be performed once or may be followed by subsequent administrations.
  • the dosage will also depend on the route of administration and will vary with the age and weight of the subject to be treated.
  • a preferred dosage of multimeric forms would be in the interval 1 mg to 70 mg per 70 kg body weight.
  • a localised or substantially localised application is preferred.
  • intranasal application is preferred.
  • the preparation further comprises pharmaceutically acceptable additives and/or carriers.
  • additives and carriers will be known in the art.
  • Administration may be a continuous infusion, such as intraventricular infusion or administration in more doses such as more times a day, daily, more times a week, weekly, etc. It is preferred that administration of the medicament is initiated before or shortly after the individual has been subjected to the factor(s) that may lead to cell death. Preferably the medicament is administered within 8 hours from the factor onset, such as within 5 hours from the factor onset. Many of the compounds exhibit a long term effect whereby administration of the compounds may be conducted with long intervals, such as 1 week or 2 weeks.
  • the administration may be continuous or in small portions based upon controlled release of the active compound(s).
  • precursors may be used to control the rate of release and/or site of release.
  • Other kinds of implants and well as oral administration may similarly be based upon controlled release and/or the use of precursors.
  • Treatment by the use of the compounds/compositions according to the invention is in one embodiment useful for inducing differentiation, modulating proliferation, stimulate regeneration, neuronal plasticity and survival of cells, for example cells being implanted or transplanted. This is particularly useful when using compounds having a long term effect.
  • the treatment may be for stimulation of survival of cells which are at risk of dying due to a variety of factors, such as traumas and injuries, acute diseases, chronic diseases and/or disorders, in particular degenerative diseases normally leading to cell death, other external factors, such as medical and/or surgical treatments and/or diagnostic methods that may cause formation of free radicals or otherwise have cytotoxic effects, such as X-rays and chemotherapy.
  • factors such as traumas and injuries, acute diseases, chronic diseases and/or disorders, in particular degenerative diseases normally leading to cell death
  • other external factors such as medical and/or surgical treatments and/or diagnostic methods that may cause formation of free radicals or otherwise have cytotoxic effects, such as X-rays and chemotherapy.
  • cytotoxic effects such as X-rays and chemotherapy.
  • the FGFR binding compounds according to the invention are useful in cancer treatment.
  • the treatment comprises treatment and/or prophylaxis of cell death in relation to diseases or conditions of the central and peripheral nervous system, such as postoperative nerve damage, traumatic nerve damage, e.g. resulting from spinal cord injury, impaired myelination of nerve fibers, postischaemic damage, e.g. resulting from a stroke, multiinfarct dementia, multiple sclerosis, nerve degeneration associated with diabetes mellitus, neuro-muscular degeneration, schizophrenia, AIz- heimer's disease, Parkinson's disease, or Huntington's disease.
  • diseases or conditions of the central and peripheral nervous system such as postoperative nerve damage, traumatic nerve damage, e.g. resulting from spinal cord injury, impaired myelination of nerve fibers, postischaemic damage, e.g. resulting from a stroke, multiinfarct dementia, multiple sclerosis, nerve degeneration associated with diabetes mellitus, neuro-muscular degeneration, schizophrenia, AIz- heimer's disease, Parkinson's disease, or Huntington'
  • the compounds according to the invention may be used for inducing differentiation, modulating proliferation, stimulate regeneration, neuronal plasticity and survival , i.e. stimulating survival.
  • the compound and/or pharmaceutical composition may be for preventing cell death of heart muscle cells, such as after acute myocardial infarction, in order to induce angiogenesis.
  • the compound and/or pharmaceutical composition is for the stimulation of the survival of heart muscle cells, such as survival after acute myocardial infarction.
  • the com- pound and/or pharmaceutical composition is for revascularisation, such as after inju- ries.
  • the present compounds are capable of stimulating angiogenesis and thereby they can promote the wound healing process.
  • the invention further discloses a use of the compound and/or pharmaceutical composition in the treatment of cancer. Regulation of activation of FGFR is important for tumor agiogenesis, proliferation and spreading.
  • a use of the compound and/or pharmaceutical composition is for the stimulation of the ability to learn and/or of the short and/or long term memory, as FGFR activity is important for differentiation of neural cells.
  • a compound and/or pharmaceutical composition of the invention is for the treatment of body damages due to alcohol consumption. Developmental malformations of foetuses, long-term neurobehavioral alterations, alcoholic liver disease are particularly concerned.
  • Therapeutic treatment of prion diseases including using a compound and/or pharmaceutical composition is still another embodiment of the invention.
  • Neoplasms such as malignant neoplasms, benign neoplasms, carcinoma in situ and neoplasms of uncertain behavior
  • cancers with dysfunction and/or over- or under-expression of specific receptors and/or expression of mutated receptors or associated with soluble receptors such as but not limited to Erb-receptors and FGF- receptors
  • diseases of endocrine glands such as diabetes mellitus I and II, pituitary gland tumor
  • psychoses such as senile and presenile organic psychotic conditions
  • alcoholic psychoses drug psychoses, transient organic psychotic conditions
  • Alzheimer's disease cerebral lipidoses
  • epilepsy general paresis [syphilis], hepatolen- ticular degeneration, Huntington's chore
  • inflammatory disease of the central nervous system such as menin- gitis, encephalitis, Cerebral degenerations such as Alzheimer's disease, Pick's disease, senile degeneration of brain, senility NOS, communicating hydrocephalus, obstructive hydrocephalus, Parkinson's disease including other extra pyramidal disease and abnormal movement disorders, spinocerebellar disease, cerebellar ataxia, Marie's Sanger-Brown, Dyssynergia cerebellaris myoclonica, primary cerebellar de- generation, such as spinal muscular atrophy, familial, juvenile, adult spinal muscular atrophy, motor neuron disease, amyotrophic lateral sclerosis, motor neuron disease, progressive bulbar palsy, pseudobulbar palsy, primary lateral sclerosis, other anterior horn cell diseases, anterior horn cell disease, unspecified, other diseases of spinal cord, syringomyelia and syringobulbia, vascular myelopathies,
  • Inflammatory and toxic neuropathy including acute infective polyneuritis, Guillain-Barre syndrome, Postinfectious polyneuritis, polyneuropathy in collagen vascular disease, disorders of the globe including disorders affecting multiple structures of eye, such as purulent endophthalmitis, diseases of the ear and mastoid process, chronic rheumatic heart disease, ischaemic heart disease, arrhythmia, diseases in the pul- monary system, respiratory system, sensoring e.g. oxygene, astma, abnormality of organs and soft tissues in newborn, including in the nerve system, complications of the administration of anesthetic or other sedation in labor and delivery, diseases in the skin including infection, insufficient circulation problem, burn injury and other mechanic and/or physical injuries.
  • Injuries including after surgery, crushing injury, burns. Injuries to nerves and spinal cord, including division of nerve, lesion in continuity (with or without open wound), traumatic neuroma (with or without open wound), traumatic transient paralysis (with or without open wound), accidental puncture or laceration during medical procedure, injury to optic nerve and pathways, op- tic nerve injury, second cranial nerve, injury to optic chiasm, injury to optic pathways, injury to visual cortex, unspecified blindness, injury to other cranial nerve(s), injury to other and unspecified nerves.
  • Scrapie Creutzfeldt-Jakob disease, Gerstmann-Straussler-Sheinker (GSS) disease; pain syndrome, encephalitis, drug/alcohol abuse, anxiety, postoperative nerve damage, peri-operative ischemia, inflammatory disorders with tissue damage, either by affecting the infections agent or protecting the tissue, HIV, hepati- tis, and following symptoms, autoimmune disorders, such as rheumatoid arthritis, SLE, ALS, and MS.
  • hyper cholestorolamia artheslerosis, disorders of amino-acid transport and metabolism, disorders of purine and pyrimidine metabolism and gout, bone disorders, such as fracture, osteoporosis, osteo arthritis (OA), Atrophic dermatitis, psoriasis, infection cased disorders, stem cell protection or maturation in vivo or in vitro.
  • bone disorders such as fracture, osteoporosis, osteo arthritis (OA), Atrophic dermatitis, psoriasis, infection cased disorders, stem cell protection or maturation in vivo or in vitro.
  • Compounds of the invention may also be used for the prevention and treatment of achondroplasia, hypochondroplasia, platyspondylic lethal skeletal dysplasia, thanatophoric dysplasia, Antley-Bixler syndrome, Apert syndrome, Beare-Stevenson syndrome, Crouzon syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, and Saethre-Chotzen syndrome.
  • a method for treatment and/or prevention of the above conditions and symptoms comprises a step of administering an effective amount of a compound and/or pharmaceutical composition to an individual in need.
  • the FGFR1 Ig1 module To study the structure and binding properties of the FGFR1 Ig1 module, we used the following recombinant proteins: the individual first and second Ig modules (Ig 1 and Ig2), and the combined second and third Ig modules (lg2-3). All recombinant proteins were properly folded as judged by NMR analysis.
  • the Ig1 and Ig2 modules of mouse FGFR1 consist of a His-tag, AGHHHHHH, and amino acids 23-119 and 140-251 , respectively (swissprot p16092).
  • the combined lg2-3 modules of mouse FGFR1 (3c isoform) consist of a His-tag, RSHHHHHH, and amino acids 141-365 (swissprot p 16092).
  • the Ig2 and lg2-3 modules were produced as previously described (Kiselyov et al., Structure 2003 11 : 691-701).
  • the Ig1 module was expressed in the KM71 strain of yeast P. pastoris (Invitrogen, USA) according to the manufacturer's instructions. All the proteins were purified by affinity chromatography using Ni 2+ -NTA resin (Qiagen, USA) and/or ion exchange chromatography and gel filtration.
  • Binding analysis was performed using a BIAcoreX instrument (Biosensor AB, Sweden) at 25 0 C using 10 mM sodium phosphate pH 7.4, 150 mM NaCI as running buffer. The flow-rate was 5 ⁇ l/min.
  • the lg2-3 modules of FGFR1 were immobilized on the sensor chip CM5 (Biosensor AB, Sweden) as previously described (Kiselyov et al., Structure 2003 11 : 691-701).
  • Binding of the Ig1 module to the immobilized lg2-3 modules was studied in the following way: A protein was injected at a specified concentration simultaneously into a flow-cell with the immobilized FGFR1 modules (Fd cell) and a control flow-cell with nothing immobilized (Fc2-cell). The curve representing unspecific binding of the protein to the surface of the Fc2-cell was subtracted from the curve representing binding of the same protein to the immobilized lg2-3 modules and the surface of the Fd -cell. The resulting curve was used for analysis.
  • NMR measurements The following samples were used for recording of NMR spectra: 2 mM Ig1 or Ig2 modules (in H 2 O or D 2 O), 2 mM 15 N-labeled Ig1 or Ig2 modules (in H 2 O), 0.5 mM 15 N 1 13 C(50%)-labeled Ig2 module (in H 2 O).
  • the buffer was 10 mM sodium phosphate pH 7.4, 150 mM NaCI, except for the double-labeled sample, where 10 mM sodium phosphate pH 7.4, 30 mM NaCI was used.
  • the following NMR spectra were recorded and used for assignment of the Ig1 and Ig2 modules: TOCSY in H 2 O or D 2 O (45 and 70 ms mixing time), NOESY in H 2 O or D 2 O (80 and 200 ms mixing time), DQFCOSY, 15 N-HSQC, 15 N-TOCSY-HSQC (70 ms mixing time), and 15 N- NOESY-HSQC (125 ms mixing time).
  • HNCACB, CBCA(CO)NH, HNCO, HN(CA)CO, HNCA, HN(CO)CA were also used. All spectra were recorded using the standard set-up provided by ProteinPack.
  • the spectra were processed by NMRPipe (Delaglio et al., J Biomol NMR 1995 6: 277- 93) and analysed by Pronto3D (Kj ⁇ er et al., Methods Enzymology 1994 239: 288- 307).
  • the NMR experiments were performed using Varian Unity Inova 750 and 800 MHz spectrometers. All spectra were recorded at 298 K.
  • FGFR-1 Trex293 cells were stably transfected with human FGFR-1 with a C- terminal Strep Il tag (IBA Biotech). The cells were maintained in the DMEM medium with Hygromycin (Invitrogen) 200 ⁇ g/ml, 10% FCS, 1 % (v/v) glutamax, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin (all from Gibco, BRL). For phosphorylation assay, 2x1 O ⁇ cells were starved overnight with medium without serum.
  • the cells were lysed by 300 ⁇ l lysis buffer with 1%(v/v) NP-40, complete protease inhibitors (Roche, Ger- many)(1 :50), and phosphatase inhibitors (Calbiochem inhibitor cocktail lll)(1 :100) in PBS. Then the protein concentration was determined using bicinchoninic acid assay (Pierce, Rockville, IL), and 500 ⁇ g proteins of each lysate was incubated with 15 ⁇ l agarose-coupled anti-phosphotyrosine antibodies (4G10-AC) (Upstate biotechnologies) for 6 hr or overnight at 4 0 C.
  • 4G10-AC agarose-coupled anti-phosphotyrosine antibodies
  • the bound proteins were washed and eluted using 180 mM phenyl phosphate (Sigma) in the chromatography columns (BIO- RAD). 25 ⁇ l of the purified protein for each sample was separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA). Im- munoblotting was performed using rabbit antibodies (diluted 1:2000) against the recombinant Strepll tag (IBA Biotech) and swine anti-rabbit IgG horseradish peroxidase conjugate (diluted 1 :2000) (DakoCytomation, Denmark) in 5% (w/v) nonfat dry milk.
  • the immune complexes were developed by SuperSignal® West Dura extended duration substrate (PIERCE), and visualized with SynGene Gene Snap ver- sion 6.00.21 software (Synoptics Ltd, UK). For all assays, the exposed bands were quantified with SynGene Gene Tool image analysis program (Synoptics Ltd, UK).
  • the solution structure of the Ig1 module was determined by NMR. A total of 1360 non-redundant NOEs was assigned and applied in the structure calculations together with 83 backbone dihedral angle constraints derived from the 3 JHNH O coupling constants. After inspection of hydrogen bond energies and the rate of hydrogen ex- change, 94 hydrogen bond constraints were applied as NOEs in the final structure calculations. A ribbon representation of the structure is shown in Fig. 1 A. The three- dimensional fold of the module belongs to the intermediate Ig subgroup and can be described as a ⁇ -barrel consisting of two ⁇ -sheets.
  • One sheet is formed by A' (L 43 - 5 V 45 ), G (G 108 -V 107 ), F (S 96 -S 105 ), C (S 63 -R 68 ) and C (V 71 -L 73 ) ⁇ -strands, and the other by A (T 26 -L 27 ), B (D 49 -R 54 ), B' (L 57 -R 58 ), E (E 85 -D 90 ) and D (T 79 -T 82 ) strands.
  • An overlay of 20 superimposed structures for the backbone atoms is shown in Fig. 1B.
  • the root mean square (RMS) deviation from the average is 1.05 A for the backbone atoms and 1.53 A for all heavy atoms. 98.8% of the ( ⁇ , ⁇ ) angle combinations of the 10 entire ensemble fall into the allowed regions of the Ramachandran plot.
  • RMS root mean square
  • the general strand topology of the Ig 1 module is similar to that of the Ig2 and Ig3 modules (Plotnikov et al., Cell 1999 98: 641-50).
  • the A/A' loop of the Ig1 module is much longer than that of the Ig2 and Ig3 modules. It contains 8 extra resi- dues compared to the Ig3 module, and 5 extra residues compared to Ig2.
  • the A/A' loop of the Ig1 module is situated perpendicularly to the ⁇ -barrel (Fig. 1C). This is noteworthy, because it is this region of the Ig1 module that forms a binding site for the Ig2 module (see below).
  • Ig1 binds to Ig2 in the area of the FGF1-lg2, heparin-lg2 and Ig2-lg2 binding sites Since Ig1 in FGFR3 binds to lg2-3 with a Kd value of 20 ⁇ M (Olsen et al., Proc Natl Acad Sci U S A 2004 101 : 935-40), it was of interest to determine this binding for FGFR1. Therefore, binding of soluble Ig1 to immobilized lg2-3 modules of FGFR1 was studied by SPR analysis. A plot of the equilibrium binding response versus the concentration of Ig1 is shown in Fig. 2A.
  • the time-course of the binding similarly to that for FGFR3, is characterized by very fast association and dissociation phases (not shown).
  • the calculated Kd value for the binding was 33 ⁇ 6 ⁇ M, which is very close to the 20 ⁇ M Kd value determined for FGFR3.
  • NMR analysis In order to identify the residues involved in the interaction between Ig1 and Ig2, NMR analysis was employed. NMR analysis provides a very sensitive method for the study of protein interactions in solution. However, it requires production of 15 N labeled proteins and assignment of their 15 N, 1 H resonance frequencies for the backbone atoms. Thus to study the Ig1-lg2 interaction by NMR, assignment of the 15 N, 1 H resonance frequencies for the backbone atoms of the Ig2 module was performed.
  • the Ig2 module contains binding sites for FGF, heparin and the Ig2 module itself (whereas the Ig3 module contains the binding site only for FGF).
  • the Ig 1 module inhibits both the FGF-FGFR and heparin-FGFR interactions (Wang et al., J Biol Chem 1995 270: 10231-5; Olsen et al., Proc Natl Acad Sci U S A 2004 101: 935-40).
  • the residues of the Ig1 module that exhibited significant perturbation (higher than 0.04 ppm) by the Ig2 module were L 27 , E 29 , Q 30 , A 31 , Q 32 , W 34 , G 35 and V 36 (Fig. 2B), and the residues of the Ig2 module that exhibited significant perturbation (higher than 0.025 ppm) by the Ig1 module were T 156 , S 157 , E 159 , K 160 , A 167 , V 168 , A 171 , K 172 , T 173 , V 174 , K 175 , S 214 , I 215 , I 216 , M 217 and S 219 (Fig. 2C).
  • the perturbed residues in the Ig1 module are located in the A/A' loop region and form a single patch, whereas the perturbed residues in the Ig2 module are located in two patches: a larger patch consisting of 12 residues (A 167 , V 168 , A 171 , K 172 , T 173 , V 174 , K 175 , S 214 , I 215 , I 216 , M 217 , S 219 ) and a smaller patch consisting of four residues (T 156 , S 157 , E 159 , K 160 ).
  • the two patches are located close to each other.
  • the primary Ig2 site binding to FGF consists of L 165 , A 167 , P 169 and V 248
  • the secondary Ig2-FGF binding site consists of P 199 , D 200 , I 203 , G 204 , G 205 , S 219 and V 221
  • the Ig2-heparin binding site consists of K 160 , K 163 , K 175 and K 177
  • the Ig2-lg2 binding site consists of A 171 , K 172 , T 173 and D 218 .
  • Fig. 3B Mapping of these binding sites onto the structure of the Ig2 module is shown in Fig. 3B.
  • a 167 from the primary and S 219 from the secondary Ig2-FGF binding sites, K 175 from the Ig2-heparin binding site, and A 171 , K 172 , T 173 from the Ig2-lg2 binding site are among the residues in the Ig2 module which are perturbed by Ig1 binding.
  • both the primary and secondary Ig2-FGF and the Ig2-heparin binding sites are adjacent to the larger Ig2 patch of perturbed residues, whereas three out of four residues of the Ig2-lg2 binding site are located within this patch.
  • Ig1 binding to Ig2 interferes with the binding of FGFR to itself, FGF and heparin, and thus that Ig1 functions as a competitive intra-molecular inhibitor of FGF-FGFR, heparin-FGFR and FGFR-FGFR interactions.
  • the Ig 1 module functions as a competitive intra-molecular inhibitor of FGF-FGFR and heparin-FGFR interactions, then given that the affinities of interactions between FGF and FGFR, heparin and FGFR, and the Ig1 and lg2-3 modules are known, it may be possible to describe quantitatively the auto-inhibitory effect of the Ig 1 module, namely: an approx. 8- and 4-fold decrease in the affinity of the FGFR1 ⁇ -FGF1 and FGFRI ⁇ -heparin interactions, respectively, as compared to the FGFR1 ⁇ -FGF1 and FGFRI ⁇ -heparin interactions (Wang ⁇ t al., J Biol Chem 1995 270: 10231-5).
  • the Ig1-lg2 linker of rat FGFRI ⁇ consists of 31 amino acids: DALPSSEDDDDDDDSSSEEKETDNTKPNRRP. Analysis of this sequence by various secondary structure prediction engines predicts a random coil for either all or most of the residues, which indicates that the linker forms a random coil in the FGFRI ⁇ molecule.
  • the random movement of the Ig 1 module around the Ig2 module is obviously restricted by the length of the linker, meaning that only a small volume around the Ig1 module is available for random movement of the Ig 1 module.
  • the real concentration of the module in the volume available to it will be used instead of its concentration in the total volume.
  • the module's concentration in a sphere (depicted as a circle in Fig. 4A) with a radius corresponding to the average distance between the N- termini of the Ig 1 and Ig2 modules will be used.
  • the average end-to-end distance of the linker region must be estimated.
  • (AR 2 ) is the root average square end-to-end distance
  • N is the number of links in the polymer
  • L is the length of the link (the angle between the consecutive links is random).
  • K 1 is the Kd value for the FGF1- FGFRI ⁇ interaction
  • [D 2 ⁇ D 1 ) is the concentration of the FGFRI ⁇ molecules in which the Ig1 and Ig2 modules are involved in the intra-molecular interaction.
  • (D 23 ⁇ D 1 ) can be determined from the following equation:
  • K 0 K 0 - N, ⁇ (l 2 +p 2 Y 2 (D 23 ) 0 , K 2 being the Kd of the interaction between
  • N A being Avogadro number
  • / being the average linker length
  • p being length of the Ig1 module.
  • the reported Kd values for the FGF-FGFR interaction range from 10 pM to 100 nM, depending on the method used.
  • the affinity of heparin-FGFR interaction is of the order of 0.3-1 ⁇ M.
  • Fig. 4B shows estimation of the binding between FGF1 and FGFRI ⁇ or FGFRI ⁇ (with linker lengths of 4.5, 6.9 and 11.3 nm) and the intra-molecular binding of the Ig1 module as a function of FGF1 concentration.
  • FGFRI ⁇ displays a substantially lower binding to FGF1 as compared to FGFRI ⁇ .
  • the Ig1 module is involved in intra-molecular binding in approx. 80% FGFRI ⁇ molecules, and, in the presence of FGF1, this percentage drops quickly with increasing concentrations of FGF1.
  • the data points from Fig. 4B were fitted to an equation describing binding with a single site (Fig. 4C).
  • linker lengths close to 3 nm are unlikely for the reasons described above, and linker lengths longer than 9-10 nm also are unlikely, because this is very close to the length of the fully extended conformation of the linker.
  • the linker length most probably ranges from 6 to 9 nm with a corresponding decrease in affinity of approximately 11- to 6-fold, which is in good agreement with the experimentally determined value of 8-fold.
  • affinity of the ligand-FGFR interaction affects the estimated inhibitory effect of the Ig1 module.
  • the inhibitory effect of the Ig1 module on the FGF-FGFRI ⁇ interaction ranges from 10.5- to 4.2-fold (Fig. 4D).
  • the Kd for the heparin-FGFR interaction is as mentioned above ca. 0.3-1 ⁇ M, and thus, the corresponding inhibitory effect of the Ig1 module is approximately 4-fold (Fig. 4D), which is in complete agreement with the experimentally determined value of 3.7-fold.
  • the quantitative analysis of the inhibitory effect of the Ig1 module presented here is in excellent agreement with the available experimental data on the kinetics of the FGF-FGFR1 and heparin-FGFR1 interactions, thereby corroborating a model in which the Ig1 module functions as a competitive intra-molecular inhibitor of the FGF- FGFR1 and FGFR1 -heparin interactions. It should be noted that since the auto- inhibitory effect of Ig1 does not depend very much on the Ig1-lg2 linker length (in the range from 4.5 to 9 nm), approximately the same auto-inhibition is expected for the other FGFR isoforms in which the Ig1-lg2 linkers are slightly shorter.
  • the stimulatory effect of the Ig2 module probably is due to inhibition of the intra-molecular Ig1-lg2 binding, which increases the number of receptor molecules in which the Ig2 modules are not blocked by intramolecular binding of Ig1 and thus available for spontaneous dimerization.
  • Addition of the Ig 1 module is expected to increase the number of receptor molecules in which the Ig2 module is bound to the Ig 1 module and, thus, decrease the number of receptor molecules available for spontaneous dimerization.
  • both peptides derived from Ig2 (FRD2a at 20, 100 and 500 ⁇ g/ml concentrations and FRD2b at 1 , 5 and 20 ⁇ g/ml concentrations) substantially increased the receptor phosphorylation similar to that of the Ig2 module, whereas the peptide derived from Ig 1 (FRDIa at 1, 5, 20, 100 and 500 ⁇ g/ml concentrations), as expected, did not have any effect.
  • D 23 stands for the lg2-3 module of the triple Ig-module FGFR (Ig1 is implied to be attached to the lg2-3 module), F - for FGF or other ligand (except Ig1), and D 1 - for
  • Cerebellar granule neurons are prepared from postnatal day seven Wistar rats largely as previously described by Drejer and Schousboe (1989) Neurochem Res. 14:751-4.
  • cerebellar tissue was dissected in modified Krebs-Ringer solution kept on ice, and treated as described for the hippocampal neurons above. All cell cultures were incubated at 37 0 C in a humidified atmosphere containing 5 % CO 2 . All animals were handled in accordance with the national guidelines for animal welfare.
  • CGN were plated at a density of 10,000 cells/cm 2 on uncoated 8-well permanox Lab- Tek chamber slides in Neurobasal medium supplemented with 0.4 % (w/v) bovine serum albumin (BSA; Sigma-Aldrich), 2 % (v/v) B27 Neurobasal supplement, 1 % (v/v) glutamax, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and 2 % 1 M HEPES (all from Gibco, BRL). Peptide solutions without or with inhibitors of various signal transduction pathways were added to a total volume of 300 ⁇ l/cm 2 , and the slides were incubated at 37 D C.
  • BSA bovine serum albumin
  • Peptide solutions without or with inhibitors of various signal transduction pathways were added to a total volume of 300 ⁇ l/cm 2 , and the slides were incubated at 37 D C.
  • the neurons were fixed with 4 % (v/v) formalde- hyde for 20 minutes and thereafter immunostained using primary rabbit antibodies against GAP-43 and Alexa Fluor secondary goat anti-rabbit antibodies.
  • Images of at least 200 neurons for each group in each individual experiment were obtained systematically by using computer assisted fluorescence microscopy as previously described (R ⁇ nn et al., 2000 op. cit). Briefly, a Nikon Diaphot inverted microscope with a Nikon Plan 2Ox objective (Nikon, Tokyo, Japan) coupled to a video camera (Grun- dig Electronics, Germany) was used for recordings. The same software package as described above for the dopaminergic neurite outgrowth assay was used to process the recorded images.
  • Figure 7 demonstrates the stimulation of neurite outgrowth of cerebellar granular neurons in response to treatment with different concentrations of the FRD2a (SEQ ID NO:8 )(A) and FRD2b (SEQ ID NO: 13) (B) peptides derived from the Ig1-to-lg2 reciprocal binding site of FGFR
  • the length of neurites in cultures is presented as a percentage of neurite length in the treated cultures compared to control (untreated cultures).
  • Both the FRD2a and FRD2b peptides are capable of stimulating neurite outgrowth.

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Abstract

La présente invention concerne de nouveaux peptides pouvant se lier à un récepteur de la famille des récepteurs du facteur de croissance des fibroblastes (FGFR) et de moduler l'activité dudit récepteur. Les peptides de l'invention sont des fragments de FGFR, lesdits fragments comprenant des résidus d'acides aminés impliqués dans l'interaction réciproque du module de type immunoglobuline 2 (Ig2) et du module de type immunoglobuline 1 (Ig1) du FGFR. L'invention concerne des séquences d'acides aminés dérivées du site de liaison réciproque d'Ig1 à lg2 du FGFR et concerne l'utilisation des peptides comprenant des séquences d'acides aminés pour le traitement de différents états pathologiques, dans lesquels les FGFR jouent un rôle proéminent. En conséquence, l'invention concerne également des compositions pharmaceutiques comprenant les composés de l'invention.
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WO2010136031A1 (fr) * 2009-05-27 2010-12-02 Københavns Universitet Peptides dérivés du récepteur du facteur de croissance des fibroblastes, qui se lient à la ncam
US20130338077A1 (en) * 2011-02-28 2013-12-19 Indiana University Research And Technology Corporation Glucocorticoid induced leucine zipper mimetics as therapeutic agents in multiple sclerosis

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010136031A1 (fr) * 2009-05-27 2010-12-02 Københavns Universitet Peptides dérivés du récepteur du facteur de croissance des fibroblastes, qui se lient à la ncam
US8637465B2 (en) 2009-05-27 2014-01-28 Kobenhavns Universitet Fibroblast growth factor receptor-derived peptides binding to NCAM
US20140234294A1 (en) * 2009-05-27 2014-08-21 Kobenhavns Universitet Fibroblast Growth Factor Receptor-Derived Peptides Binding to NCAM
US20130338077A1 (en) * 2011-02-28 2013-12-19 Indiana University Research And Technology Corporation Glucocorticoid induced leucine zipper mimetics as therapeutic agents in multiple sclerosis
US8999937B2 (en) * 2011-02-28 2015-04-07 Indiana University Research And Technology Corporation Glucocorticoid induced leucine zipper mimetics as therapeutic agents in multiple sclerosis

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